Enzyme stabilization and thermotolerance function of the intrinsically disordered LEA2 proteins from date palm

In date palm, the LEA2 genes are of abundance with sixty-two members that are nearly all ubiquitous. However, their functions and interactions with potential target molecules are largely unexplored. In this study, five date palm LEA2 genes, PdLEA2.2, PdLEA2.3, PdLEA2.4, PdLEA2.6, and PdLEA2.7 were cloned, sequenced, and three of them, PdLEA2.2, PdLEA2.3, and PdLEA2.4 were functionally characterized for their effects on the thermostability of two distinct enzymes, lactate dehydrogenase (LDH) and β-glucosidase (bglG) in vitro. Overall, PdLEA2.3 and PdLEA2.4 were moderately hydrophilic, PdLEA2.7 was slightly hydrophobic, and PdLEA2.2 and PdLEA2.6 were neither. Sequence and structure prediction indicated the presence of a stretch of hydrophobic residues near the N-terminus that could potentially form a transmembrane helix in PdLEA2.2, PdLEA2.4, PdLEA2.6 and PdLEA2.7. In addition to the transmembrane helix, secondary and tertiary structures prediction showed the presence of a disordered region followed by a stacked β-sheet region in all the PdLEA2 proteins. Moreover, three purified recombinant PdLEA2 proteins were produced in vitro, and their presence in the LDH enzymatic reaction enhanced the activity and reduced the aggregate formation of LDH under the heat stress. In the bglG enzymatic assays, PdLEA2 proteins further displayed their capacity to preserve and stabilize the bglG enzymatic activity.

Plants have evolved complex regulatory pathways to counter the effects of adverse climatic conditions. The mechanisms to improve abiotic stress tolerance are largely dependent on protein molecules that directly function and regulate various plant physiological processes and signaling pathways. The late embryogenesis abundant (LEA) protein gene family is a group of functional proteins that protect and reduce plant cell damages under abiotic stress conditions 1 . These proteins are disordered in their structure and characterized by repeated motifs 2 . Based on the conserved amino acid sequence motifs, LEA proteins were classified into eight distinct groups, with LEA2 proteins being the most predominant group in plants. Primarily, LEA2 proteins were found in large quantities in mature seeds of Gossypium hirsutum 3 , and were ubiquitously expressed in flowering and non-flowering plants 4 . They mainly accumulate in the late phases of seed development and in vegetative tissues for plant responses to environmental constraints 5 . LEA2 proteins are immensely hydrophilic and characterized as intrinsically disordered proteins (IDPs) that can vary their conformation in response to the changes in the ambient microenvironment 6 . Among the LEA2 proteins sub-family, dehydrins (DHNs) are a well-known biochemical group that is mainly composed of high proportion of charged and polar amino acids, and low fraction of hydrophobic and non-polar residues 7 .
LEA2 proteins are broadly involved in plants physiological responses to improve abiotic stress tolerance. Overexpression of Triticum aestivum L., TaLEA2-1 in wheat enhanced their root growth, plant height, and led to higher catalase activity in comparison to the wild type seedlings. TaLEA2 www.nature.com/scientificreports/ in TaLEA2-1 transgenic wheat plants 5 . Furthermore, in a recent study, LEA2 gene, PtrDHN-3, from Populus trichocarpa was found to play an essential role in salt and drought stress tolerance8. It was observed that the overexpression of PtrDHN-3 increased the salt tolerance of transgenic yeast, and improved the germination rate, fresh weight, and chlorophyll content of transgenic Arabidopsis thaliana plants under salt stress 8 . In addition, the Arabidopsis plants transformed with cotton LEA2 genes displayed higher growth under drought stress compared to the wild type 9 . Moreover, in maize a KS-type DHN gene, ZmDHN13, was isolated and its overexpression in transgenic tobacco plants enhanced the oxidative stress tolerance 10 . In another study, overexpression of Prunus mume LEA2 gene in transformed tobacco and Escherichia coli improved cold stress tolerance 11 . Furthermore, overexpression of two A. thaliana DREs, AtDREB1A or AtDREB2A, resulted in an induction of cold stress interlinked genes of LEA2 proteins, such as the rd29A and COR47 12 . In the putative promoters of LEA2 genes of upland cotton, G. hirsutum, several abiotic stress-related cis-elements were found. It included MYBCORE, ABRELATERD1, ABRE-like sequence and ACG TAT ERD1 elements which are known to have a functional role in abiotic stresses 13,14 . The presence of these stress promoter elements strongly supports the role of LEA2 proteins in enhancing abiotic stress tolerance in plants growing under hostile climates.
In the resurrection plant, Craterostigma plantagineum, several LEA mRNA transcripts and proteins have been identified during a cycle of desiccation stress 15 . Similarly, to gain insights into the Phoenix dactylifera, a woody extremophile plant that thrives under harsh environmental conditions, an RNA-Seq analysis was performed to understand the date palm ABA signaling pathway in response to drought stress 16 . Date palm's pinnae was treated with the hormone ABA that resulted in the expression of 153 differentially expressed genes (DEGs) 16 . Among the highlighted genes, LEA genes were reported to be upregulated along with phosphatases from the PP2C family, ATP binding cassette (ABC) transporters, and the guard cell transcription factor MYB74. In addition, a whole genome sequencing of date palm was performed and LEA2 genes were found abundantly present in date palm's genome assembly 17 . It was found that LEA2 genes consisted of sixty-two variants in date palm 17 . Moreover, high expression of LEA2 genes were reported in date palm inoculated with Piriformospora indica under salinity stress 18 . These findings argue in favor of LEA2 genes involvement in the date palm's abiotic stress tolerance mechanisms.
Although several molecular and physiological analyses of stress related genes in P. dactylifera have been performed, the functional characterization of P. dactylifera LEA2 (PdLEA2) genes is still obscure 19 . However, it is believed that LEA2 proteins are highly flexible unstructured proteins that are able to function as chaperones and interact with several partner molecules such as proteins, membranes, nucleic acids, and metal ions 20 . A recent study annotated the functional properties of LEA2 proteins, which included their roles in protecting membranes, stabilizing macromolecules, supporting in free radical scavenging, and acting as antioxidants for alleviating the oxidative damages caused to plants under the abiotic stress conditions 6 . Similar to LEA2 proteins, major heatshock proteins (hsp) have some kind of related roles in solving the problem of misfolding and aggregation, as well as their role as chaperones. Thus, LEA2 proteins play crucial roles in protecting other protein molecules and enzymes from aggregation and stabilize their activities under several hostile treatment conditions. Nevertheless, few functional enzymatic assays have been developed for examining the protective role of LEA2 proteins on enzyme activities. Moreover, it is observed that majority of the industrial enzymes are degraded due to the extreme temperature in the processing conditions, during which the enzyme needs to be preserved.
Based on the functional properties of LEA2 proteins, they can be utilized for the stabilization and preservation of enzymatic activities from heat during industrial processes for a longer period. In relation to this, it is necessary to investigate the role of LEA2 proteins in preserving the thermosensitivity of lactate dehydrogenase (LDH) and β-glucosidase (bglG) enzymes. LDH enzyme acts as a redox cofactor by utilizing the NADH/NAD + pair for catalyzing the interconversion of pyruvate (oxo acid) and lactate (alpha-hydroxy acid) 21 . It is used for the conversion of plant pyruvate into lactic acid under anaerobic conditions. While bglG is a cellulolytic enzyme that is employed in the degradation of cellulosic biomass. It is involved in the hydrolysis of carbohydrates such as starch, glycogen, and their disaccharides derivatives into their monomers 22 . Both of these enzymes are used on a large scale for industrial and biotechnological applications.
Thus, the objective of the current study was to examine and characterize the role of P. dactylifera LEA2 proteins on enzymes thermotolerance. The present study emphasized on the similarities and dissimilarities between the five PdLEA2 proteins in terms of their physicochemical characteristics, amino acid constituents, disorder propensity, preserved structural motifs, and predicted their secondary and tertiary structure. Furthermore, PdLEA2 recombinant proteins were produced and their protective role on LDH and bglG enzymatic activities and stabilities under heat stress condition was elucidated. The findings of the present study will be the first breakthrough to identify the chaperone property of date palm LEA2 proteins in protein-enzyme interaction. It will build a pathway for identification of LEA2 proteins integral partners and target components in the cell, providing expanded perception into their protective phenomenon for crop stress tolerance.

Results
1Isolation and physicochemical analysis of PdLEA2 sequences. The PdLEA2 genes were mapped to different chromosomes in date palm. Based on a BLAST search against the Barhee BC4 date palm genome assembly (GCF_009389715.1), PdLEA2.2 was not assigned to any chromosome, while the PdLEA2. 3,PdLEA2.4,PdLEA2.6,and PdLEA2.7 were distributed in the chromosomes 7, 14, 3, and 2, respectively. The mRNA sequences were deposited in NCBI GenBank and the mRNA and protein accessions are provided in Table S1.
The PdLEA2 proteins predicted molecular weights ranged from 22 Structural characteristics of PdLEA2 proteins. Secondary structure and disorder propensity. A high similarity was identified in the secondary structure and disorder propensity of the five PdLEA2 proteins. The prediction of PdLEA2 sequence composition secondary structure was performed using PSIPRED ( Fig. 1B; Table 2). Evidently, the most predominant folded secondary structure is the β-strand, 38-49%, predicted with the highest level of confidence. Random coils were present and distributed throughout the entire sequence. However, a long stretch of coiled region was predicted at the N-terminal region of all PdLEA2 proteins, which increased the total composition of this state, and ranged between 31-45% of the structures. As LEA2 proteins are known to harbor intrinsically disordered regions, disorder propensity of PdLEA2 was predicted using DISOPRED. In all the PdLEA2 sequences, a disordered region of varying length was identified at the N-terminal region of the protein (Fig. 1A). Additionally, a disordered region was also identified near the C-terminal region of PdLEA2.3, PdLEA2.4, and PdLEA2.7. The disordered regions corresponding to PdLEA2 proteins are enclosed in black rectangles in Fig. 1B.
Three-dimensional structural model of PdLEA2 proteins. 3D structures of PdLEA2 proteins were modelled using AlphaFold2, an artificial intelligence-based protein structure prediction system. The predicted structures of the five proteins are shown in Fig. 1C. As evident from the figures, in all proteins, the N-terminal region had a distinct disordered region for which a 3D structure could not be predicted. This is indicated by the low value of predicted local distance difference test (pLDDT) value, an indicator of the residue-level confidence in the predicted structure, in this region. Overall, structurally all five PdLEA2 proteins appear to have a conserved architecture consisting of a disordered N-terminal region, followed by an α-helix and a tertiary structure consisting of two stacked β-sheets. Notably, the stacked β-sheet structure is duplicated in the case of PdLEA2.3.
Transmembrane region. An overlapping region with high hydrophobicity (Fig. 1B) and helicity ( Fig. 1A) was predicted in each of the PdLEA2 proteins following the predicted disordered region (Fig. 1B). Therefore, to assess if the PdLEA2 proteins could localize to the membrane, multiple transmembrane domain prediction tools-MEMSTAT, DeepTMHMM and TOPCONS-were used to predict transmembrane regions in PdLEA2 (Table 2). Interestingly, all of these predictors indicated that the hydrophobic helix identified above is potentially a transmembrane α-helix. The only discrepancy appears to be in PdLEA2.3, where there was no consensus between the results of the three predictors. Furthermore, the predictors suggested that the disordered N-terminal region of PdLEA2 proteins is intracellular, while the β-strand rich region resides outside the cell.
Conserved domains and protein motifs. Conserved domains in the PdLEA2 sequences were identified using CD-Search against NCBI's Conserved Domain Database (CDD). PdLEA2.2, PdLEA2.4, PdLEA2.6 and PdLEA2.7 proteins were found to harbor one complete conserved domain of the LEA_2 superfamily. While, PdLEA2.3 was observed to have two Water Stress and Hypersensitive response (WHy) domain, which is also a member of the LEA_2 superfamily (Table 3). All identified conserved domains (pfam03168 and smart00769) are members of the cl12118 LEA_2 superfamily. Additionally, MEME identified the presence of 4 statistically significant conserved motifs within PdLEA2 sequences (Table 4). However, only one conserved motif with a consensus sequence of DVLIRNPN was shared by all the five sequences.
Phylogenetic analysis and multiple alignment. Global multiple sequence alignment of the PdLEA2 protein sequences produced low sequence identity ranging between 8.28-26.41%, indicating poor overall sequence conservation characteristic of the LEA2 superfamily. However, these sequence differences do not restrict it from producing a similar three-dimensional fold as discussed earlier. A search of all date palm protein sequences in NCBI RefSeq protein database, harboring a member of the LEA superfamily sequence, showed that most of these sequences have neither been well characterized, nor annotated appropriately. Needless, a phylogenetic tree    Fig. 2A-B). However, in roots under control or salinity stress condition, PdLEA2 genes expression showed no significant difference between the tolerant and the sensitive varieties ( Fig. 2A). In contrast, in leaves under salinity stress, a significant difference was observed between the PdLEA2 genes expression level between the tolerant  www.nature.com/scientificreports/ and the sensitive varieties, but not under the control condition (Fig. 2B). The PdLEA2 genes level of expression were higher in leaves than in roots for both genotypes.

Production and purification of recombinant PdLEA2 proteins. The PdLEA2.2, PdLEA2.3, and
PdLEA2.4 ORFs were cloned in frame with the polyhistidine tag of the pET28a expression vector. Recombinant PdLEA2 proteins were expressed in E. coli cells (BL21 strain) and assessed by SDS-PAGE. After the induction with IPTG, PdLEA2 proteins accumulated in high amounts in the E. coli cells ( Fig. 3A; Fig. S2). The affinity chromatography with nickel column was used to purify the overexpressed PdLEA2 proteins. The purity of PdLEA2 proteins was verified through Western blot analysis using an anti-His6 antibody ( Fig. 3B; Fig. S2). As expected, the immunoblot revealed a band for the PdLEA2 proteins, but not with the control.
PdLEA2 proteins stabilization of the LDH enzyme under heat stress conditions. The ability of PdLEA2.2, PdLEA2.3 and PdLEA2.4 to inhibit the LDH activity loss after heat stress was tested. The effects of PdLEA2.2, PdLEA2.3 and PdLEA2.4 proteins were compared with the BSA, a non-specific protectant, and with buffer treated LDH enzyme without adding protein ( Fig. 4A-C). After 10 min of heat stress, at the mass ratios of 1:1, 1:20, and 1:40, a significant difference (p < 0.001) was observed in the stabilization of LDH enzyme with the PdLEA2 proteins, compared to BSA and buffer without additional protein. It was found that PdLEA2 proteins provided a greater shield to LDH than BSA and buffer, with the highest protection observed for the PdLEA2.2, 90% at 1:1 and 115% at 1:40 within 10 min of the heat stress (Fig. 4A). It was observed that half of LDH enzyme www.nature.com/scientificreports/ activity was lost with buffer following 10 min of heating at 50 °C. Similarly, after 20 min of heat stress, there was also a significant difference (p < 0.001) between the LDH enzymatic activity with PdLEA2 proteins, compared to BSA, and the buffer without addition of proteins at the three mass ratios (Fig. 4B). The percentage of enzymatic activity recovery was the highest for the PdLEA2.2 protein that augmented with increasing mass ratio of proteins, 65% (1:1) and 115% (1:40). Furthermore, a significant difference (p < 0.001) was observed within 30 min of enzyme activity under heat stress (Fig. 4C) between PdLEA2 proteins recovery activity, BSA and buffer without proteins at mass ratio of 1:1 and 1:20. However, at the mass ratio of 1:40 and after 30 min of heat stress, there was a significant difference (p < 0.001) between the PdLEA2 proteins and buffer without proteins, but no significant difference was observed between the stabilization provided by the PdLEA2 proteins and BSA. At the highest mass ratio (1:40) and after 30 min at 50 °C, PdLEA2.2 protected at least 90% of the enzymatic activity, while PdLEA2.3 and PdLEA2.4 preserved 86% and 84% of the enzyme activity, respectively. Whereas the recovery of enzyme activity was reduced to 80% for the BSA and to 20% for the buffer without protein. This indicated that the LEA2 proteins provided the stabilization of enzyme activity with no effects of the presence of a second nonspecific protein at the different mass ratios with longer incubation times. Thus, it was observed that the enzymatic activity of LDH was completely protected after 10 min, 20 min or 30 min of heat stress condition with the presence of PdLEA2 proteins at the mass ratios of 1:1, 1:20, and 1:40.

PdLEA2 proteins inhibition of LDH aggregation under heat stress treatments. LDH enzyme
forms aggregate when exposed to dehydration, heating, or freeze-thaw treatments. This study examined the capability of PdLEA2 proteins to decrease the aggregation of LDH enzyme under heat stress conditions through measuring the apparent light scattering absorbance of proteins solutions. The impact of PdLEA2 proteins on the enzymes was studied at two mass ratios of 1:1 and 1:2. It was observed that LDH formed massive aggregation after heating at 80 °C for 20 min. At both the mass ratios, there was a significant difference (p < 0.001) in the inhibition of LDH aggregate formation under the heat stress with addition of PdLEA2 proteins and BSA (Fig. 5). The existence of the PdLEA2 proteins declined the enzymatic aggregation of LDH at both the mass ratios in contrast to the BSA (Fig. 5). For enzymatic assay under heat stress, the absorbance in the presence of PdLEA2 was lowered more than the half of the aggregate formation in the presence of BSA in both the mass ratios (Fig. 5). It was found that LDH aggregation was consistently lower at a similar activity rate between the three PdLEA2 proteins.
PdLEA2 improved the thermostability and activity of bglG. To test the protective effect of PdLEA2 proteins on bglG enzymatic activity at 70 °C, recombinant PdLEA2.2, PdLEA2.3 and PdLEA2.4 proteins (0.5 µg ml −1 ) were added to the reaction of the bglG enzyme. The treatments were performed with and without PdLEA2 proteins for 15 min time intervals up to 90 min. A significant difference (p < 0.001) was observed in the enzymatic activity with and without PdLEA2 proteins at the different time intervals of heat stress reaction (Fig. 6). The bglG enzyme activity decreased drastically in the absence of PdLEA2 proteins within 30 min to 22%, while it was found that PdLEA2.2 protein preserved 90% of the enzyme activity at the same time interval. In

Discussion
In plants, LEA2 proteins have been characterized functionally and are involved in responses to the environmental stresses of various plants, specifically drought-tolerant plants 23 , halophytes 24 , resurrection plants 15 , and cold tolerant plants 25 . In this study, LEA2 proteins from P. dactylifera were identified, characterized, and explored for the first time for their potential implication on enzyme stability and thermotolerance.
The LEA2 genes are abundantly present in the P. dactylifera genome assembly, having sixty-two members, in comparison to fifty-two and forty-six in rice and sorghum, respectively 17 . In addition, there were thirty LEA2 genes in Prunus salicina 26 , twenty-seven in Solanum lycopersicum 27 , fifty-three in populus 28 , twenty-nine in Solanum tuberosum 29 and thirty-two in tea plant 4 . In contrast, the number of LEA2 genes was much higher in Ramonda serbica 20 and Arachis hypogaea 30 , which had 127 and 78 members, respectively. The abundance of LEA2 Figure 5. LDH anti-aggregation activity of PdLEA2 under heating stress. Enzymatic aggregation was monitored in spectrophotometer at the absorbance rate of 340 nm. PdLEA2 proteins and BSA were added to the LDH enzymatic reaction at two different mass ratios. Data are the means ± SD (n = 3), with different letters indicating a significant difference (p < 0.05) analyzed using Tukey's HSD test after one-way ANOVA. www.nature.com/scientificreports/ genes in date palm can be depicted as being the last members to evolve among the LEA genes group or because of the whole gene duplication event within this group as it occurred in cotton plants 9 . The high abundance or redundancy of a particular gene group indicates the major part it plays in improving the plants survival. Date palm growing in the arid regions continuously faces harsh environmental conditions. Their growth under the environmental stresses can be attributed to the accumulation of diverse stress tolerant LEA2 genes or due to the integration of these genes with several other gene regulatory mechanisms for activating adaptive responses. The location of a gene on the chromosome plays an essential role in shaping an organism's trait and its evolution. The PdLEA2 genes were broadly distributed and located on different chromosomes in the date palm genome. The range of amino acid sequence of the identified PdLEA2 proteins were between 202 to 317 aa, which is larger than found in bay beans 31 and tea plants 32 , but is in close range to Rhododendron catawbiense DHNs, RcDhn 1-5 33 . However, the molecular weight of PdLEA2 proteins were smaller than the A. thaliana 34 and cotton9, which were extending between 67.2 to 160.7 kDa, respectively. The pI values of PdLEA2 proteins ranged between 4.85 to 9.96, indicating that the isolated PdLEA2 proteins were basic in nature, similar to Brachypodium distachyon LEA2 proteins 35 and wheat LEA proteins 36 . The average pI of the PdLEA2 proteins displayed higher association with the G. hirsutum LEA groups, specifically DHNs and SMPs9.
In terms of hydrophobicity, GRAVY values indicated that two of the proteins, PdLEA2.3 and PdLEA2.4, were moderately hydrophilic, while PdLEA2.7 was hydrophobic and PdLEA2.2 and PdLEA2.6 were in between. Notably, Kyte-Doolittle plots indicated the presence of a long stretch of hydrophobic region which was predicted to form a transmembrane helix. The moderate hydrophilic nature of PdLEA2.3 and PdLEA2.4 proteins can enable them to form a hydrogen bond with water molecules, thereby get dissolved in water, which is a thermodynamically favored interaction. The hydrophilic property was previously observed in LEA2 proteins of other plants 37 . It allows them to get totally or partially disordered, a unique feature of the LEA proteins. The hydrophilic property is further required to form flexible structural elements, such as molecular chaperones, that are essential for the plant's protection against desiccation 38 . On the other hand, the hydrophobic nature of the PdLEA2.7 proteins enables them to fold spontaneously into complex structures and further allow the discharge of nonpolar amino acids from the solvents. This attribute commonly occurs in the water channel proteins such as aquaporins (AQPs), which are highly hydrophobic and play a significant part in drought and salinity stress tolerance of plants 39 . Instability index showed that most of the isolated PdLEA2 proteins were unstable in contrast to that of Sorghum bicolor LEA, SbLEA and S. lycopersicum LEA, SiLEAs that were similar to stable PdLEA2.3 protein 27,40 . PdLEA2 proteins displayed high aliphatic index suggesting their relative volume is occupied largely by aliphatic side chains such as alanine, isoleucine, leucine, and valine that enhances their thermostability. LEA proteins are not transmembrane proteins as they can be located in nucleus, chloroplast, mitochondria, and cytoplasm 41 . Contrarily, PdLEA2 proteins exhibited transmembrane helices, which indicates their expression in subcellular compartments. The presence of at least one transmembrane α-helix was similarly identified in R. serbica LEA2 proteins20.
The conserved domain analysis of PdLEA2 proteins revealed the presence of a LEA_2 superfamily domain, pfam03168, in PdLEA2.2, PdLEA2.4, and PdLEA2.7. This LEA_2 domain-containing proteins have been correlated with various plant tolerance responses against several abiotic stresses such as heat, drought, salinity, osmotic stress, UV damage, and oxidative stress [42][43][44][45] . Furthermore, PdLEA2.3 consisted of WHy domain and LEA_2 superfamily, smart00769. The presence of WHy domain sequence has been found as an ORF in certain bacterial genomes of phylum Firmicutes 46 . It has been reported that the recombinant bacterial WHy protein exhibited stress tolerance phenotype in E. coli and provided protection to protein denaturation in vitro 46 . Protein denaturation implies the damage of the tertiary structure, resulting in the loss of stability and structure of the protein. Moreover, the PdLEA2.4 and PdLEA2.6 had the LEA_2 superfamily domain, cl12118, which was also identified in cotton LEA2 proteins 9 .
The majority of LEA2 proteins have typical motifs that are essential for their identification. In PdLEA2 proteins, four distinctive motifs were found to be present. The motif length varied between 8 and 24 amino acids. The motif 2 was occurring in all the PdLEA2 proteins, whereas the motif 1 was present in PdLEA2.2 and PdLEA2.4 proteins only. Similarly, in PdLEA2.2, PdLEA2.4, and PdLEA2.6 only conserved motif 3 was present. In addition, motif 4 was found in PdLEA2.2, PdLEA2.4 and PdLEA2.7. The presence of a common motif composition indicates identical functional specificity within the PdLEA2 proteins subgroup as identified in S. tuberosum 47 . Identical results of LEA proteins group-specific conserved motifs were reported earlier for Arabidopsis 34 , S. lycopersicum 27 , Prunus 26 , poplar28, maize 48 , Brassica 49 , and cotton 9 .
The LEA2 proteins are predicted to be IDPs 6 . The secondary and 3D structural analysis of all five PdLEA2 proteins displayed a disordered region at the N-terminal site. This region was followed by an α-helix and a tertiary structure consisting of three β-hairpins and β-strands. Thus, the most predominant folded secondary structure was the β-strand, with random coils distributed throughout PdLEA2 protein sequences. The structure of PdLEA2 proteins were similar to the secondary structure prediction in bay bean and R. serbica LEA proteins in relation to β-sheet, α-helix, and random coil content 31 . However, this is in contradiction with the wheat LEA proteins that had lower β-sheet strands 36 .
Due to the absence of validation and good annotation for LEA2 proteins, the phylogenetic tree generated using date palm protein sequences harboring members of the LEA2 superfamily showed that, in most cases, PdLEA2 proteins were located next to proteins annotated as NDR1/HIN1-like in the date palm genome. NDR1/ HIN1-like (NHL) gene groups include the Harpin-induced gene 1 (HIN1) and Nonrace-specific disease resistance gene 1 (NDR1) 50 . HIN1 gets accumulated by a harpin protein that is involved in different plant defense responses to abiotic stresses50. Whereas the cloning of NDR1 gene from A. thaliana indicated its responsive function to plant disease resistance 51 .
LEA2 genes are largely expressed in response to abiotic stress in plants 6 . In this study, PdLEA2 genes displayed a relatively higher expression in leaves of the tolerant and sensitive varieties, Lulu and khalas seedlings after Scientific Reports | (2023) 13:11878 | https://doi.org/10.1038/s41598-023-38426-w www.nature.com/scientificreports/ salinity stress treatments, respectively. This finding is consistent with the expression of LEA genes in cotton, S. bicolor, and maize plants vegetative tissues 9,40,48 . Leaves are the major plant parts that are inflicted by the salinity stress. The increased accumulation of PdLEA2 transcripts in leaf tissues indicates their role in preserving the structural integrity and preventing the salinity damage to the membranes. Furthermore, leaves are major site for photosynthesis, which get affected due to the excess release of reactive oxygen species (ROS) under salt stress 52 . ROS are detrimental to plant growth and high PdLEA2 genes expression in the leaves, indicate their involvement in the scavenging of the ROS through the enhancement of the activities of antioxidant enzymes, similar to Oryza sativa LEA5 gene 53 . Protein denaturation is a frequent physiological phenomenon occurring in plant cells faced with abiotic stress. Different factors affect the enzymatic activities such as the temperature, pH, substrate and enzyme concentrations, and the presence or absence of inhibitors and activators in the reaction. Thermal stabilization of enzyme activity such as Glucose oxidase (GOD) has been reported in the presence of trehalose, a most widely known compatible solute acting as a chemical chaperone 54 . In the present study, it was found that PdLEA2 proteins protected the LDH and bglG enzymatic activities from the damages of heating stress during their reaction conditions. The presence of PdLEA2 proteins allowed higher LDH and bglG enzymatic activities recovery under heat stress, indicating that the isolated PdLEA2 proteins have strong ability to protect the enzymes. The enzymatic protection of the LEA2 proteins was more efficient than provided by BSA. This can be attributed to the disorder structure, transmembrane helix, and folded region consisting of β-sheets of PdLEA2 proteins during the heat stress or due to their ability to act as chaperones and bind to membranes, metal ions and water. The LDH and bglG loss of catalytic activity was not only preserved, but PdLEA2 proteins further prevented the aggregation of LDH enzyme. Particularly during drying, decrease in aggregation, has been found for other LEA proteins from plants, which was associated to the hypothesis of molecular shield 55 . Several studies have indicated that DHNs can protect LDH and bglG enzyme activities against the damage caused by various stresses. In relation to PdLEA2 proteins, it was found that CdDHN4-L and CdDHN4-S recovered the LDH enzymatic activities during free-thaw damage and heating stress 56 . In addition, it was reported in the presence of Picea Wilsonii DHN, PicW1, LDH enzyme activity was higher than the blank control and BSA at 43 °C to 55°C 57 . Alternatively, the conditions of heating and freezing stress causes water stress and certain DHNs are found to play the role of antiaggregant agents of the other protein or enzyme molecules under this stress 58 . The function of PdLEA2 proteins in protecting enzyme activities during heat stress was similar to the wheat LEA3 protein, TdLEA355. Indeed, the addition of TdLEA3 at the highest mass ratio in the reaction, preserved 90% of the LDH enzymatic activity at 48 °C after 30 min. The structural domains of LEA2 proteins and the mechanism controlling the enzymatic activity protection under stress conditions, has been investigated in few studies as a heat protection mechanism of LEA2 proteins. In Brassica napus, LEA3 proteins protected LDH under desiccation stress, which was attributed to its hydrophilic nature, β-sheets, α-helix, and random coil propensity 38 . In the present study, PdLEA2.2, PdLEA2.3 and PdLEA2.4 were moderately hydrophilic, had β-sheets and random coil propensity, which may have contributed to the protection of LDH enzyme under the heat stress. Moreover, in cryoprotection assay, formation of random coil occurred in Arabidopsis COR15 59 , which indicated the role of random coil structure in the enzymes cryoprotection. Few studies have been conducted in investigating the role of LEA2 proteins on protecting the bglG enzyme under heat stress. In our previous study, we found that DHN-5 from wheat played a relevant role in protecting the enzyme bglG against heat stress 60 . Truncation assay of DHN-5 indicated that K-segments were vital to the thermal protection of LDH and bglG 61 . It was observed that the truncated forms of DHN-5 that contained only one or two K-segments were able to protect LDH and bglG enzymatic activities against the damages caused by various stresses in vitro albeit to lesser extent than the wild-type protein. Beside the impact of K-segments in improving the LDH and bglG heat stability, these truncated forms properly refolded the enzymes after heat stress as the wild-type protein. However, the PdLEA2 proteins are lacking any K-segment and protected the bglG enzyme activity, indicating a possible role of hydrophilicity and β-sheets structure in protecting the bglG enzyme under heat stress.

Conclusion
In this study, LEA2 proteins from date palm were characterized, and the functional analysis elucidated their roles in protecting enzymes. The PdLEA2 proteins had high sequence similarity, hydrophilicity, and were disordered in structure. It was found that the isolated PdLEA2 proteins from date palm protected the enzymatic stability and activity of LDH and bglG enzymes under heat stress. The protective role of PdLEA2 proteins can be due to their disorder structure, transmembrane helix, and folded region consisting of β-sheets, which enables them to stabilize various partner molecules such as enzymes or target proteins in different cellular compartments during the heat stress. The opulence of LEA2 genes in date palm and their functional characterization sets a major foundation for further research in understanding the evolutionary relation of LEA2 gene family and their potential role in plants tolerance to abiotic stress conditions. In addition, the generation of transgenic plants overexpressing PdLEA2 genes will provide protection against environmental constraints such as salinity, drought, and heat stresses. Moreover, the use of fermentation for the outgrowth of bacterial cells at a large volume to enhance the production of recombinant PdLEA2 proteins is considered as a potential application from this study. The present findings reinforce the proposition that PdLEA2 proteins are vital molecules that can be harnessed as molecular chaperones for developing novel recombinant thermoresistant enzymes with improved rate of reaction and specificity. www.nature.com/scientificreports/ volume that were rehydrated with the introduction of 14 μl of buffer. The impact of heating was tested with the sample's treatment to 50 °C for up to 60 min. The enzyme activity was determined by adding one ml of freshly assembled assay buffer (at pH 7.4 of 10 mM Na 3 PO 4 , with 2 mM NADH, and 10 mM pyruvic acid) into the samples of LDH enzyme. At 340 nm, NADH oxidation was observed for 3 min, during which a linear rate of reaction was present. The enzyme activity was calculated using the rate of absorbance decrease (ΔOD/min) × 8095 = U l −1 .
The assay samples were analyzed in triplicates. The effect of PdLEA2 proteins on the thermostability of the bglG enzyme was tested by using purified PdLEA2 proteins at optimal concentration of 0.5 µg ml −1 as additive in the bglG assay for two different temperatures, 50 °C as the optimal temperature and 70 °C during which its catalytic efficiency is lost drastically. The enzyme bglG from Aspergillus niger was obtained from Megazyme and used according to the manufacturer's instructions. Thermal stability of bglG was measured through the incubation of the purified enzyme at the required temperatures for 30 min intervals, using 1 mM of para-nitrophenyl β-D-glucopyranoside as substrate, followed by measuring the relative activity. The reaction was stopped with the addition of 0.6 ml of 0.4 M Glycine-NaOH buffer (pH 10.8), and the released p-nitrophenol was evaluated at 400 nm. 18,000 M −1 cm −1 was used as the molecular extinction coefficient of p-nitrophenol. One unit of enzymatic activity was determined as the quantity of bglG needed to liberate one mol of p-nitrophenol under the assay condition per min.
Bioinformatics analysis of PdLEA2 sequences and structure. Various physical and chemical parameters of the PdLEA2 proteins, including molecular mass, theoretical pI, instability index, aliphatic index, and grand average of hydropathy (GRAVY), were assessed using Exapsy ProtParam 62 . Hydropathy analysis of the PdLEA2 protein sequences was performed using Expasy ProtScale based on the Kyte & Doolittle scale62. Disordered regions in the protein sequences were analyzed using a CS-BLAST against the Database of Disordered Protein Predictions (D 2 P 2 ) 63 , which aggregates results from several disorder prediction tools and DISOPRED 64 . Conserved domains were analyzed using NCBI's CD-Search tool and protein motifs of PdLEA2 sequences were identified using Multiple EM for Motif Elicitation (MEME) 65 . Secondary structure prediction was performed using PSIPRED 66 and transmembrane (TM) regions were predicted using MEMSTAT 67 , DeepTMHMM 68 and TOPCONS 69 . Models of the three-dimensional structure of PdLEA2 sequences were generated using an inhouse installation of AlphaFold2 70 .
To generate a phylogenetic tree of PdLEA2 proteins, all available P. dactylifera LEA2 protein sequences were obtained from NCBI RefSeq Protein database. The presence of LEA2 superfamily was confirmed in these sequences using NCBI's CD-Search tool. The obtained and the PdLEA2 sequences reported here were aligned using MAFFT version 4.790 71 in Geneious Prime 2022.2.2 (https:// www. genei ous. com). A phylogenetic tree was generated using EBI's Simple Phylogeny tool using the neighbor joining method. The tree was visualized, annotated, and rendered using Interactive Tree of Life v5 72 .
Statistical analysis. Statistical parameters such as mean and standard deviation (SD) were calculated for the PdLEA2 genes expression level and enzymatic activities of LDH and bglG under heat stress condition with different PdLEA2 protein treatments. Three biological replicates were used for each of the enzymatic assays and PdLEA2 expression analysis. Data were analyzed using one-way ANOVA with Tukey's HSD test for the evaluation of significant differences between PdLEA2 proteins and control treatments under heat stress. Each of the variable's normality and homoscedasticity were evaluated. The analyses were performed using the R statistical software.